Membrane fusion is a common stage of diverse cell biological processes, including exocytosis and protein trafficking, fertilization and entry of enveloped viruses into host cells. Even for the best-characterized fusion mediated by influenza virus hemagglutinin (HA) the mechanism remains unknown. At the time of fusion, membranes are packed with fusogenic proteins. Does each of these proteins serve as an independent fusion machine? Or do adjacent individual proteins interact with each other in the plane of the membrane? Are proteins outside of the contact zone involved in fusion? To address these questions experimentally and to characterize the role of HA-HA interactions, we investigated the effects of the surface density of HA on efficiency of HA activation and fusion. Based on the results of these studies and analysis of the literature we have proposed a new mechanism of protein-mediated fusion. The work can be divided into 3 projects. 1. Synchronized activation and unfolding of HAs in multimeric fusion machine. It has been hypothesized that fusion is mediated by a multiprotein machine that somehow delivers the conformational energy of multiple proteins to the intermediates of membrane fusion. If this is correct, how do these proteins synchronize their refolding to minimize dissipation of the energy? We found that triggering of the conformational change in an individual HA trimer is affected by the proximity of other HAs. We modified the surface density of HA of Japan and X31 strains of influenza and assayed the transition of HA from its initial to its low pH conformation both as the development of HA susceptibility to S-S reduction and as the digestion of the exposed fusion peptide by thermolysin. Conformational change in HA was also detected functionally as inactivation of HA by low pH pretreatment in the absence of a target membrane. As expected, Japan HA-membranes retained fusogenic activity after longer low pH incubations than did X31 HA-membranes. Our results suggest that this difference reflects slow activation, rather than inactivation as formerly thought. More importantly, we show that in both slow- and fast-activating strains, the percentage of activated HA increases with the increase in HA density, indicating that HA activation involves positive inter-trimer cooperativity. We propose that this spreading of the activation among adjacent HA trimers leads to the synchronized release of their conformational energy and is the mechanism by which multiple fusion proteins coordinate their activity at the fusion site. 2. Reversible stages of the low-pH-triggered restructuring of HA. The refolding of HA at the pH of fusion has been often considered to be a concerted and irreversible discharge of a loaded spring, with no distinct intermediates between the initial and final conformations. However, in our new study, we found that HA refolding involves reversible conformations with a lifetime of minutes. After reneutralization, low pH-activated HA returns from the conformations, wherein both the fusion peptide and the kinked loop of the HA2 subunit are exposed, but the HA1 subunits have not yet dissociated, to a structure indistinguishable from the initial one in functional, biochemical, and immunological characteristics. The rate of the transition from reversible conformations to irreversible refolding depends on pH and on the presence of target membrane. Importantly, recovery of the initial conformation is blocked by the interactions between adjacent HA trimers. To explain the positive cooperativity of HA activation at low pH, we hypothesized that individual HAs first establish a reversible activated conformation. Our new work experimentally confirms this prediction and identifies an early reversible form of low-pH-activated HA from which HA can revert to the initial conformation, if there are no adjacent trimers to interact with. Inter-trimer interaction shifts HA restructuring towards irreversible stages. We propose that the existence of this relatively long-lived intermediate state before the major refolding of HA is of importance for coupling between this refolding and fusion. We hypothesize that at low pH HA starts to flicker between its initial form and an early ?primed? reversible state, with most of the time spent in the initial form. The delay before the discharge of most of the HA conformational energy gives the adjacent activated HAs in the contact region time to interact and to synchronize their discharge. While pathways of diverse membrane fusion reactions appear to have common membrane intermediates, the structures of the specialized fusion proteins can be rather dissimilar. However reversible stages of refolding of fusion protein identified in our work for the fusion protein of influenza virus have been discussed in the literature for some other viruses including tick-borne encephalitis, rabies virus, and HIV. By analogy with HA-mediated fusion, we hypothesize that different viral fusion reactions and intracellular fusion involve a distinct reversible stage of refolding of fusogenic proteins that allows adjacent trigger-activated proteins to assemble at the contact site. Subsequent concerted discharge of most of the conformational energy of these proteins drives membrane fusion. 3. The protein coat in membrane fusion: lessons from fission. Multiple biological processes involve two opposite rearrangements of membrane configuration referred to as fusion and fission. While membrane intermediates in protein-mediated fusion have been studied in some detail, the global force, which drives sequential stages of fusion reaction from early local intermediates to an expanding fusion pore, remains unknown. Neither of the published hypothetical mechanisms of protein-mediated fusion has addressed the question how fusion proteins generate the membrane tension necessary for expansion of the fusion pore. A local protein machine is unable to generate tension in a membrane part larger than the initial fusion site. To generate a membrane stress driving fusion pore expansion, the fusion machine must act on a large area of the membrane. This consideration along with the established role of interactions between low pH-activated HA in fusion has motivated us to search for a mechanism which would be based on fusion protein aggregation. Fusion proceeds via stages, which are analogous but oppositely directed to membrane budding-off/fission driven by protein coats. The energy needed for membrane budding and for fission of the membrane neck in the best-characterized budding-fission reactions including intracellular fission and exit of enveloped viruses from host cells is apparently produced by self-assembly of the coat proteins at the membrane surface. On the basis of the analogy between fusion and fission, we propose that an interconnected coat formed by membrane-bound activated fusion proteins surrounding the membrane contact zone generates the driving force for fusion. This fusion protein coat has a strongly curved intrinsic shape opposite to that of the protein coat driving fission. Since the rigidity of the protein coat is likely much greater than that of the lipid bilayer, to adopt the intrinsic shape and thus to relieve the internal elastic stresses, the protein coat spontaneously bends out of the initial shape of the membrane surface. This bending produces elastic stresses in the underlying lipid bilayer and drives its fusion with the apposing membrane. The hypothesis that fusion proteins outside of the contact zone participate in the generation of the driving force for fusion offers new interpretation for a number of known features of fusion reaction mediated by the prototype fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other fusion reactions.